Division of normal cells is controlled by complex interactions between factors telling the cell to divide and other factors telling the cell to stop dividing. Controlling the steps of cell division, collectively known as the cell cycle, is a complex interplay between promoter (oncogenes) and suppressor genes.
Cancer is a disease resulting from uncontrolled cell division caused by mutations in promoter (oncogenes) and/or suppressor genes. In cancer cells, oncogene products are over expressed and tumor suppressor gene products are lost. Initial evidence for the existence of tumor suppressor genes emerged from studies of chromosomal deletions in familial cancer syndromes. Deletions in these same regions are also often observed in sporadic cancers. The restoration of a single copy of a missing or altered tumor suppressor gene will often re-establish control over the growth of cancerous cell lines and suppress tumor formation in animal cancer models.
Many genes and their products with positive effects on cell proliferation such as growth factors and their cognate receptors, transcription factors, and cyclins and cyclin dependent kinases have been identified and extensively studied. These represent oncogenes whose aberrant overexpression leads to uncontrolled growth. Genes involved in cell cycle arrest have been more difficult to isolate and characterize because of their recessive nature. These tumor suppressors or negative regulators of cell proliferation exhibit loss of function in tumors.
Tumor suppressor genes code for negative regulators that suppress the proliferation of cells and are of immense interest because of their importance in understanding normal and cancerous cell growth. The prohibitin 3′ untranslated region (3′ UTR) falls into a major category of tumor suppressors that are inhibitors of DNA synthesis. The cDNA coding for prohibitin was originally identified and cloned in a screen to discover senescence regulating mRNAs highly expressed in normal compared to regenerating rat liver (McClung, et al 1989 “Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver,” Biochem Biophys Res Comm 164.1316-1322; and Nuell, et al 1991 “Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells,” Mol Cell Bio 11.1372-1381).
The human prohibitin gene maps to chromosome 17 at q21 near BRCA1, and two alleles (designated “B” and “non-B”) have been described. (Jupe, et al. 1995 “Prohibitin antiproliferative activity and a lack of heterozygosity in immortalized cell lines,” Exp Cell Res 218.577-580, Jupe et al 1996 “The 3′ untranslated region of prohibitin and cellular immortalization,” Exp Cell Res 224:128-135, Jupe, et al. 1996 “Prohibitin in breast cancer cell lines loss of antiproliferative activity is linked to 3′ untranslated region mutations,” Cell Growth and Differentiation 7:871-878; and White, et al. 1991 “Assignment of the human prohibitin gene (PHB) to chromosome 17 and identification of a DNA polymorphism,” Genomics 11.228-230). In both human and rat, the prohibitin gene has six introns and seven exons (Altus, et al 1995 “Regions of evolutionary conservation between rat and human prohibitin-encoding genes,” Gene 158:291-294) and produces two transcripts, one apparently 1.2 and the other 1.9 kb in length, as defined by Northern blotting experiments (Nuell, et al 1991 Mol Cell Bio 11 1372-1381, Jupe, et al 1995. Exp Cell Res 218.577-580, Jupe et al 1996. Exp Cell Res 224.128-135; and Jupe, et al 1996 Cell Growth and Differentiation 7 871-878). Both transcripts code for the same 30,000 Dalton protein, and in mammalian tissues, the level of protein expression generally parallels the level of total message (Nuell, et al. 1991. Mol Cell Bio 11:1372-1381; Jupe, et al 1995 Exp Cell Res 218 577-580, Jupe et al 1996 Exp Cell Res 224 128-135, Jupe, et al 1996 Cell Growth and Differentiation 7 871-878, White, et al 1991 Genomics 11 228-230, Altus, et al 1995 Gene 158 291-294, and McClung, et al 1995 “Prohibitin, potential role in senescence, development, and tumor suppression,” Exp Gerontol 30 99-124). In addition to rat and human, prohibitin has been cloned from mouse (Terashima, et al 1994 “The IgM antigen receptor of B lymphocytesis associated with prohibitin and a prohibitin-related protein,” EMBO J 13 3782-3792), yeast (Franklin, D. S. and Jazwinski, S M. 1993. “A yeast homolog of the rat prohibitin gene is differentially expressed and determines longevity in Saccharomyces cerevisiae, J Cell Biochem Suppl 17D 159), Drosophila (Eveleth, D. D J and Marsh, J. L 1986. “Sequence and expression of the Cc gene, a member of the dopa decarboxylase gene cluster of Drosophila, possible translational regulation,” Nucleic Acids Res, 14 6169-6183), and Pneumocystis carinii (Narasimhan, et al. 1997. “Prohibitin, a putative negative control element present in Pneumocystis carinii,” Infection and Immunity 65 5125-5130). The protein is highly conserved throughout evolution, and the deduced amino acid sequences of human and rat prohibitin are identical except for one conservative amino acid change (McClung, et al 1995. Exp Gerontol 30.99-124, and Sato, et al. 1992 “The human prohibitin gene located on chromosome 17q21 is mutated in sporadic breast cancer,” Cancer Res 52 1643-1646). The majority of the protein is localized to the mitochondria, and roles in diverse processes such as cellular aging in yeast (Jazwinski, S. M. 1996 “Longevity, genes, and aging,” Science 273:54-59, and Coates, et al. 1997. “The prohibitin family of proteins regulate replicative lifespan,” Current Biology 7:R607-R610), development and viability in Drosophila (Eveleth, D. D J. and Marsh, J L 1986 Nucleic Acids Res, 14 6169-6183), and granulosa cell proliferation in mammals (Thompson, et al 1997 “Steroidogenic acute regulatory (StAR) protein (p25) and prohibitin (p28) from cultured rat ovarian granulosa cells,” J Reproduct Fertility 109.337-348) have been reported. In yeast and Pneumocystis, prohibitin protein has been shown to have a possible role in the ras signalling pathway (Narasimhan, et al 1997. Infection and Immunity 65 5125-5130, and Jazwinski, S M 1996 Science 273 54-59). Prohibitin has been shown to interact with retinoblastoma tumor suppressor proteins (Rb) in vivo and in vitro and was effective in repressing E2F-mediated transcription, while a prohibitin mutant could not bind to Rb, repress E2F activity, or inhibit cell proliferation (Wang, et l 1999 “Prohibitin, a potential tumor suppressor, interacts with RB and regulates E2F function,” Oncogene 18:3501-3510).
Prohibitin gene structure and function have been examined in eleven immortalized human cell lines which have been classified into four complementation groups (A-D) (Jupe, et al. 1995 Exp Cell Res 218.577-580; and Jupe et al. 1996. Exp Cell Res 224 128-135). Human breast cancer cell lines have also been examined because the gene is located at 17q21, a chromosomal region that frequently undergoes loss of heterozygosity (LOH) in sporadic and familial breast cancers (Nagai, et al 1994 “Detailed deletion mapping of chromosome segment 17q12-21 in sporadic breast tumors,” Genes, Chromosomes, and Cancer 11:58-62). Cell proliferation assays performed following the introduction of full length (19 kb) wild type prohibitin RNA showed that normal human diploid fibroblasts (HDF), 75% of breast cancer cell lines examined, and all four Group B cell lines (but no cell lines from any of the other groups) were inhibited at the G1-S transition in the cell cycle (Jupe, et al. 1995. Exp Cell Res 218.577-580, Jupe et al 1996. Exp Cell Res 224.128-135; and Jupe, et al 1996 Cell Growth and Differentiation 7 871-878). Surprisingly, sequence analysis of the prohibitin gene showed that the 3′ UTR from prohibitin sensitive cancer cell lines differed from wild type at one or more bases, while the 3′ UTR from insensitive lines exhibited the wild type sequence. There were no coding region sequence alterations in any of the cell lines. (Jupe, et al 1995. Exp Cell Res 218:577-580, Jupe et al. 1996 Exp Cell Res 224:128-135; and Jupe, et al. 1996. Cell Growth and Differentiation 7.871-878). These findings suggest that the loss of growth control in the cancer cell is due to mutations in the prohibitin 3′ UTR.
International Application No. WO 96/40919 published Dec. 19, 1996, disclosed that mutations in the 3′ UTR of the B type allele are diagnostic for increased susceptibility to breast cancer and that reintroduction of either a portion or entire normal 3′ UTR of the prohibitin gene into early stage tumors can be employed as a therapeutic agent for treatment of cancer.
International Application No. WO 98/20167 published May 14, 1998, disclosed a method for determining a patient's susceptibility to breast cancer by identifying the patient's germline genotype at position 729 in the prohibitin 3′ UTR (SEQ ID NO 2) from either genomic DNA or RNA transcribed from genomic DNA which contains the 3′ UTR of the prohibitin gene.
International Application No. WO 99/24614 published May 20, 1999, disclosed a method for determining a patient's susceptibility to other types of cancer besides breast cancer (e.g., prostate or ovarian cancer) by identifying the patient's germline genotype at position 729 in the prohibitin 3′ UTR (SEQ ID NO:2) from either genomic DNA or RNA transcribed from genomic DNA which contains the 3′ UTR of the prohibitin gene.
U.S. Pat. No. 5,776,738 issued Jul. 7, 1998 and U.S. Pat. No. 5,922,852 issued Jul. 13, 1999 disclosed a purified nucleic acid fragment consisting of a portion of the 3′ UTR region of the prohibitin gene which can be used in determining a patient's susceptibility to breast cancer and other cancers.
It has now been found that the introduction of single stranded oligonucleotides, preferably DNA from the 3′ UTR of wild-type prohibitin or RNA transcribed therefrom, or synthetically manufactured oligonucleotides of like sequence, most preferably RNA transcribed from the wild type prohibitin 3′ UTR, into three breast cancer cell lines leads to arrested cell proliferation, while RNA transcribed from mutated prohibitin 3′ UTR has no antiproliferative activity. Thus, growth control is reestablished when wild-type prohibitin RNA is introduced. Whereas cellular proliferation assays have previously demonstrated cell cycle arrests following the introduction of genes coding for p53 or p21 cyclin dependent kinase inhibitor (i.e., WAF1, SD11, CAP20 or CIP1) and subsequent production of functional protein, it has now been found that wild type RNAs transcribed from portions of the prohibitin 3′ UTR (or other oligonucleotides as described herein) can be directly administered as therapeutic agents against cancer, thus bypassing the requirements of the more “traditional” gene therapy approaches where a protein-producing gene is introduced into the cell or chromosome.